Tumour markers

ABSTRACT

A method of determining the immune response of a mammal to circulating tumour marker proteins is described in which a sample of bodily fluid, for example plasma or serum, is contacted with a panel of two or more distinct tumour marker antigens. The presence of complexes between the tumour marker antigens and any autoantibodies to the antigens present in the sample are detected and provide an indication of an immune response to a circulating tumour marker protein. The method is useful for the diagnosis of cancer, particularly for identifying new or recurrent cancer in an otherwise assymptomatic patient.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §120 or 35 U.S.C.§365(c) of PCT International application PCT/GB99/01479, designatingGreat Britain, and filed May 11, 1999. PCT application PCT/GB99/01479,of which this application is a national stage filing under 35 U.S.C.§371, was published under PCT Article 21(2) in English.

Foreign priority benefits are claimed under 35 U.S.C. §119(a)-(d) or 35U.S.C. §365(b) of Great Britain application number 9810040.7, filed 11May 1998, which designated at least one country other than the UnitedStates.

The invention relates to methods of detecting or quantitativelymeasuring the immune response of a mammal to circulating tumour markersor tumour markers expressed on the surface of tumour cells, also totumour marker antigens for use in these methods, to kits for performingthe methods and to the use of these methods in the detection of cancer,in monitoring the progress of cancer, in detecting recurrent disease incancer patients who have previously undergone anti-cancer treatment andin predicting the response of a cancer patient to a particular course oftreatment.

The development and progression of cancer in a patient is generallyfound to be associated with the presence of markers in the bodily fluidof the patient, these “tumour markers” reflecting different aspects ofthe biology of the cancer (see Fateh-Maghadam, A. & Steilber, P. (1993)Sensible use of tumour markers. Published by Verlag GMBH, ISBN3-926725-07-9). Tumour markers are often found to be altered forms ofthe wild type proteins expressed by ‘normal’ cells, in which case thealteration may be a change in primary amino acid sequence, a change insecondary, tertiary or quaternary structure or a change inpost-translational modification, for example, abnormal glycosylation.Alternatively, wild type proteins which are up-regulated orover-expressed in tumour cells, possibly as a result of geneamplification or abnormal transcriptional regulation, may also be tumourmarkers.

Established assays for tumour markers present in bodily fluids tend tofocus on the detection of tumour markers which reflect tumour bulk andas such are of value late in the disease process, for example, in thediagnosis of metastatic disease. The most widely used of these markersinclude carcinoembryonic antigen (CEA) and the glycoprotein termed CA15.3, both of which have been useful mainly as indicators of systemicdisease burden and of relapse following therapy (Molina, R., Zanon, G.,Filella, X. et al. Use of serial carcinoembryonic antigen and CA 15.3assays in detecting relapses in breast cancer patients. (1995) BreastCancer Res Treat 36: 41-48) These markers are of limited use earlier inthe disease progression, for example in the screening of asymptomaticpatients. Thus, in the search for tumour markers present in bodily fluidthat are of use earlier in the disease process the present inventorshave sought to identify markers which do not depend on tumour bulk perse.

Differences between a wild type protein expressed by ‘normal’ cells anda corresponding tumour marker protein may, in some instances, lead tothe tumour marker protein being recognised by an individual's immunesystem as ‘non-self’ and thus eliciting an immune response in thatindividual. This may be a humoral (i.e B cell-mediated) immune responseleading to the production of autoantibodies immunologically specific tothe tumour marker protein. Autoantibodies are naturally occurringantibodies directed to an antigen which an individual's immune systemrecognises as foreign even though that antigen actually originated inthe individual. They may be present in the circulation as circulatingfree autoantibodies or in the form of circulating immune complexesconsisting of autoantibodies bound to their target tumour markerprotein.

As an alternative to the direct measurement or detection of tumourmarker protein in bodily fluids, assays could be developed to measurethe immune response of the individual to the presence of tumour markerprotein in terms of autoantibody production. Such assays wouldessentially constitute indirect detection of the presence of tumourmarker protein. Because of the nature of the immune response, it islikely that autoantibodies can be elicited by a very small amount ofcirculating tumour marker protein and indirect methods which rely ondetecting the immune response to tumour markers will consequently bemore sensitive than methods for the direct measurement of tumour markersin bodily fluids. Assay methods based on the detection of autoantibodiesmay therefore be of particular value early in the disease process andpossibly also in relation to screening of asymptomatic patients, forexample to identify individuals “at risk” of developing disease.

Tumour marker proteins observed to elicit serum autoantibodies include aparticular class of mutant p53 protein, described in U.S. Pat. No.5,652,115, which can be defined by its ability to bind to the 70 kd heatshock protein (hsp70). p53 autoantibodies can be detected in patientswith a number of different benign and malignant conditions (described inU.S. Pat. No. 5,652,115) but are in each case present in only a subsetof patients. For example, one study utilizing an ELISA assay fordetection of autoantibodies directed against the p53 protein in theserum of breast cancer patients reported that p53 autoantibodies wereproduced by 26% of patients and 1.3% of control subjects (Mudenda, B.,Green, J. A., Green, B. et al. The relationship between serum p53autoantibodies and characteristics of human breast cancer. (1994) Br JCancer 69: 4445-4449.). A second tumour marker protein known to elicitserum autoantibodies is the epithelial mucin MUC1 (Hinoda, Y. et al.(1993) Immunol Lett. 35: 163-168; Kotera, Y. et al. (1994) Cancer Res.54: 2856-2860).

In most cancers resulting from a progressive accumulation of geneticalterations, such as breast cancer, the presence of tumour markers inbodily fluids reflects the development and progression of disease but nosingle marker on its own summates all clinically important parameters.For example, the characteristics of a marker useful for diagnosis ofcancer may be quite different from markers which convey informationabout prognosis. Furthermore, in each clinical situation (i.e. diagnosisor prognosis) different markers may be required when dealing withprimary cancer and secondary (metastatic) cancer and a different markeragain may be required to provide a method of measuring the effectivenessof a particular course of treatment. Different clinical situationstherefore require different biological markers and, as has been observedwith p53, not all patients express the same set of tumour markerproteins. It is therefore difficult to envisage any one single tumourmarker being universally applicable to all patients in all stages ofdisease.

It is an object of the present invention to provide an improved assaysystem for the detection of bodily fluids-borne tumour markers which ismore generally useful in all patients and in a variety of differentclinical situations.

Accordingly, in a first aspect the invention provides a method ofdetecting the immune response of a mammal to circulating tumour markerproteins or tumour cells expressing said tumour marker proteins, whichmethod comprises steps of:

-   -   (a) contacting a sample of bodily fluids from said mammal with a        panel of two or more distinct tumour marker antigens;    -   (b) determining the presence or absence of complexes of said        tumour marker antigens bound to autoantibodies present in said        sample of bodily fluids, said autoantibodies being        immunologically specific to said tumour marker proteins.        whereby the presence of said complexes is indicative of the        immune response to circulating tumour marker proteins or tumour        cells expressing said tumour marker proteins.

The method of the invention, which may be hereinafter referred to as a‘panel assay’, utilises a panel of two or more tumour marker antigens tomonitor the overall immune response of an individual to a tumour orother carcinogenic/neoplastic change. The method thus providesessentially a ‘profile’ of the immune response for that individual,indicating which tumour markers elicit an immune response resulting inautoantibody production. The method of the invention is preferred forthe detection of an immune response resulting in the production ofcirculating free autoantibodies.

Because the assay method of the invention performed on a sample ofbodily fluids taken from the patient it is essentially non-invasive andcan be repeated as often as is thought necessary to build up a profileof the patient's immune response throughout the course of disease. Asused herein the term ‘bodily fluids’ includes plasma, serum, wholeblood, urine, sweat, lymph, faeces, cerebrospinal fluid or nippleaspirate. The type of bodily fluid used may vary depending upon the typeof cancer involved and the use that the assay is being put to. Ingeneral, it is preferred to perform the method on samples of serum orplasma.

As will be illustrated in the Examples given below, the use of a panelof two or more tumour marker antigens to monitor autoantibody productionis more sensitive than the use of single markers and gives a much lowerfrequency of false negative results. The actual steps of detectingautoantibodies in a sample of bodily fluids may be performed inaccordance with immunological assay techniques known per se in the art.Examples of suitable techniques include ELISA, radioimmunoassays and thelike. In general terms, such assays use an antigen which may beimmobilised on a solid support. A sample to be tested is brought intocontact with the antigen and if autoantibodies specific to the tumourmarker protein are present in the sample they will immunologically reactwith the antigen to form autoantibody-antigen complexes which may thenbe detected or quantitatively measured. Detection ofautoantibody-antigen complexes is preferably carried out using asecondary anti-human immunoglobulin antibody, typically anti-IgG oranti-human IgM, which recognise general features common to all humanIgGs or IgMs, respectively. The secondary antibody is usually conjugatedto an enzyme such as, for example, horseradish peroxidase (HRP) so thatdetection of autoantibody/antigen/secondary antibody complexes isachieved by the addition of an enzyme substrate and subsequentcalorimetric, chemiluminescent or fluorescent detection of the enzymaticreaction products.

The panel assay of the invention uses a panel of tumour marker-relatedantigens. The panel may be tailored to detect a particular cancer, or acancer at a particular stage of development. The tumour marker antigensmay be wild type or mutant tumour marker proteins isolated from samplesof biological fluid from normal individuals or from cancer patients orfrom cell lines expressing the tumour marker protein or they may be fulllength recombinant tumour marker proteins, viral oncogenic forms oftumour marker proteins or antigenic fragments of any of theaforementioned proteins. The term ‘antigenic fragment’ as used hereinmeans a fragment which is capable of eliciting an immune response.

The panel assay may be performed in a multi-well format in which eachone of the two or more antigens is placed in a separate well of amulti-well assay plate or, alternatively, in a single-pot format inwhich the entire panel of antigens is placed in a single container. Thepanel assay may be performed in a qualitative format in which theobjective is simply detection of the presence or absence ofautoantibodies or in a quantitative format which provides a quantitativemeasurement of the amount of autoantibodies present in a sample.

Preferred markers for inclusion into the panel of tumour marker antigensinclude the epidermal growth factor receptor-related protein c-erbB2(Dsouza, B. et al. (1993) Oncogene. 8: 1797-1806), the glycoprotein MUC1(Batra, S. K. et al. (1992) Int. J. Pancreatology. 12: 271-283) and thesignal transduction/cell cycle regulatory proteins Myc (Blackwood, E. M.et al. (1994) Molecular Biology of the Cell 5: 597-609), p53(Matlashewski, G. et al. (1984) EMBO J. 3: 3257-3262; Wolf, D. et al.(1985) Mol. Cell. Biol. 5: 1887-1893) and ras (or Ras) (Capella, G. etal. (1991) Environ Health Perspectives. 93: 125-131), including theviral oncogenic forms of ras which can be used as antigens to detectanti-ras autoantibodies, and also BRCA1 (Scully, R. et al. (1997) PNAS94: 5605-10), BRCA2 (Sharan, S. K. et al. (1997) Nature. 386: 804-810),APC (Su, L. K. et al. (1993) Cancer Res. 53: 2728-2731; Munemitsu, S. etal. (1995) PNAS 92: 3046-50), CA125 (Nouwen, E. J. et al. (1990)Differentiation. 45: 192-8) and PSA (Rosenberg, R. S. et al. (1998)Biochem Biophys Res Commun. 248: 935-939). As aforementioned, the assayscan be formed using tumour marker antigens which are forms of theseproteins isolated from human bodily fluids or from cultured cells orantigenic fragments thereof or full length or truncated recombinantproteins or antigenic fragments thereof.

Preferably the tumour marker antigens are labelled with biotin so thatthey can easily be attached to a solid support, such as a multi-wellassay plate, by means of the biotin/avidin or biotin/streptavidininteraction. Tumour marker antigens labelled with biotin may be referredto herein as ‘biotinylated’ proteins. To facilitate the production ofbiotinylated tumour marker antigens for use in the assay methods of theinvention, cDNAs encoding a full length recombinant tumour markerprotein, a truncated version thereof or an antigenic fragment thereofmay be expressed as a fusion protein labelled with a protein orpolypeptide tag to which the biotin co-factor may be attached via anenzymatic reaction. A useful system for the expression of biotinylatedfusion proteins is the Pinpoint™ system supplied by Promega Corporation,Madison Wis., USA. The present inventors have surprisingly found thatwith the use of biotinylated tumour marker antigens as antigens they areable to detect autoantibodies in a much higher percentage of patientsthan is observed using non-biotinylated antigen.

The assay method of the invention may be employed in a variety ofdifferent clinical situations such as, for example, in the detection ofprimary or secondary (metastatic) cancer, in screening for earlyneoplastic or early carcinogenic change in asymptomatic patients oridentification of individuals ‘at risk’ of developing cancer(particularly breast cancer, bladder cancer, colorectal cancer orprostate cancer) in a population or asymptomatic individuals, in thedetection of recurrent disease in a patient previously diagnosed ascarrying tumour cells who has undergone treatment to reduce the numberof tumour cells or in predicting the response of an individual withcancer to a course of anti-cancer treatment.

The assay method of the invention is suitable for detection of manydifferent types of cancer, of which examples are breast, bladder,colorectal, prostate and ovarian. The assay of the invention maycomplement existing methods of screening and surveillance. For examplein the case of primary breast cancer it could be used to alertclinicians to biopsy small lesions on mammograms which radiographicallydo not appear suspicious or to carry out breast imaging or to repeatimaging earlier than planned. In the clinic, the assay method of theinvention is expected to be more objective and reproducible compared tocurrent imaging techniques (i.e. mammography and ultrasound), thesuccess of which can be operator-dependent.

As aforesaid the panel of tumour marker antigens may be tailored havingregard to the particular application. A panel of at least p53 andc-erbB2 is particularly useful for many types of cancer and canoptionally be supplemented with other markers having a known associationwith the particular cancer, or a stage of the particular cancer, to bedetected. For example for breast cancer the panel might include MUC 1and/or c-myc and/or BRCA1 and/or BRCA2 and/or PSA whereas bladder cancerthe panel might optionally include MUC 1 and/or c-myc, for colorectalcancer ras and/or APC for prostate cancer PSA and/or BRCA 1 or forovarian cancer BRCA1 and/or CA125. There are other preferred embodimentsin which p53 or c-erbB2 are not necessarily essential. For example, inthe case of breast cancer suitable panels could be selected from thefollowing:

p53 and MUC 1 with optional c-erbB2 and/or c-myc, and/or BRCA1 and/orBRCA2 and/or PSA;

p53 and c-myc with optional c-erbB2 and/or MUC1 and/or BRCA1 and/orBRCA2 and/or PSA;

p53 and BRCA1 with optional c-erB2 and/or MUC 1 and/or c-myc and/orBRCA2 and/or PSA;

p53 and BRCA2 with optional c-erbB2 and/or MUC 1 and/or c-myc and/orBRCA1 and/or PSA;

c-erbB2 and MUC 1 with optional p53 and/or c-myc, and/or BRCA1 and/orBRCA2 and/or PSA;

c-erbB2 and c-myc with optional p53 and/or MUC1 and/or BRCA1 and/orBRCA2 and/or PSA;

c-erbB2 and BRCA1 with optional p53 and/or MUC 1 and/or c-myc and/orBRCA2 and/or PSA;

c-erbB2 and BRCA2 with optional p53 and/or MUC 1 and/or c-myc and/orBRCA1 and/or PSA;

In the case of colorectal cancer suitable panels could be selected fromthe following:

p53 and ras with optional c-erbB2 and/or APC;

p53 and APC with optional c-erbB2 and/or Ras;

Ras and APC with optional p53 and/or c-erbB2

In the case of prostate cancer suitable panels could be selected fromthe following:

p53 and PSA with optional BRCA1 and/or c-erbB2;

c-erbB2 and PSA with optional p53 and/or BRCA1.

In the case of ovarian cancer suitable panels could be selected from thefollowing:

p53 and CA125 with optional c-erbB2 and/or BRCA1;

c-erbB2 and CA125 with optional p53 and/or BRCA1.

In a second aspect the invention provides a method of determining theimmune response of a patient to two or more circulating tumour markerproteins or to tumour cells expressing said tumour marker proteins andidentifying which one of said two or more tumour marker proteins elicitsthe strongest immune response in the patient, the method comprisingcontacting a sample of bodily fluids from said patient with a panel oftwo or more distinct tumour marker antigens, measuring the amount ofcomplexes formed by binding of each of said tumour marker antigens toautoantibodies present in the sample of bodily fluids, saidautoantibodies being immunologically specific to said tumour markerproteins and using the measurement obtained as an indicator of therelative strength of the immune response to each tumour marker proteinand thereby identifying which one of said two or more tumour markerproteins elicits the strongest immune response in the patient.

The assay described above, which may be hereinafter referred to as a‘selection assay’ is useful in the selection of a course of vaccinetreatment wherein the single tumour marker protein identified aseliciting the strongest immune response or a combination of markerseliciting strong immune response is/are used as the basis of ananti-cancer vaccine treatment.

Preferred tumour marker antigens for use in the selection assay are anyof the tumour marker antigens mentioned above and preferably theantigens are labelled with biotin. The actual steps of detectingautoantibodies in a sample of bodily fluids may be performed inaccordance with known immunological assay techniques, as described abovefor the panel assay.

The invention also provides methods for the detection or quantitativemeasurement of the immune response of a mammal to a circulating tumourmarker protein or tumour cells expressing the tumour marker proteinwherein the tumour marker protein is MUC1, c-erbB2, Ras, c-myc, BRCA1,BRCA2, PSA, APC, CA125 or p53, the method comprising the steps ofcontacting a sample of bodily fluids from the mammal with the tumourmarker antigen and determining the presence or absence of complexes ofthe tumour marker antigen bound to autoantibodies immunologicallyspecific to the tumour marker protein or antigenic fragment thereof,whereby the presence of said complexes is indicative of the immuneresponse to said circulating tumour marker protein or tumour cellsexpressing the tumour marker protein.

The assays described above, which may be hereinafter referred to as‘single marker assays’, use a single type of tumour marker as antigenrather than using a panel of two or more tumour markers. The singlemarker assays may be used in any clinical situation, for example,screening for early neoplastic or carcinogenic change in asymptomaticpatients, identification of individuals ‘at risk’ of developing cancer,early diagnosis and early detection of recurrence in a patientpreviously diagnosed as carrying tumour cells which patient hasundergone treatment to reduce the number of said tumour cells or inpredicting the response of a patient to a course of anti-cancertreatment, including surgery, radiotherapy, immune therapy, vaccinationetc.

The single marker assays are particularly useful in situations where thetumour marker eliciting the strongest immune response in a given patienthas been previously identified, possibly using the selection assaydescribed above. For example, in a situation in which an initialselection assay has been performed to establish which tumour markerelicits the strongest immune response in a given patient, subsequentfollow-up, detection of recurrence or monitoring of treatment may becarried out using a single marker assay to only detect or measureautoantibodies to that tumour marker previously identified as elicitinga strong immune response in that patient.

The actual steps of detecting autoantibodies in a sample of bodilyfluids may be performed in accordance with known immunological assaytechniques, as described above for the panel assay. Preferably thetumour marker protein used as antigen is labelled with biotin so that itmay be easily attached to a solid support by means of the biotin/avidinor biotin/streptavidin interaction.

In a further aspect, the present invention provides a preparationcomprising a human MUC1 protein which MUC1 protein manifests all theantigenic characteristics of a MUC1 protein obtainable from the bodilyfluids of a patient with advanced breast cancer.

Preferably the MUC1 protein exhibits altered affinity for the antibodiesB55, C595, BC4W154, DF3, B27.29, 115D8, 27.1, SM3, Ma552, HMPV and BC2compared to MUC1 protein isolated from normal human urine. Mostpreferably the MUC1 protein is isolated from the serum of one or morehuman patients with advanced breast cancer. This can be accomplishedusing the protocol given in the Examples listed herein.

As will be described in detail in Example 2, the present inventors havefound immunological differences between MUC1 isolated from normalindividuals and MUC1 isolated from patients with advanced breast cancer.Possibly as a result of these differences, the inventors have found thatthe MUC1 protein isolated from serum of patients with advanced breastcancer (hereinafter referred to as ABC MUC1) is more sensitive when usedas antigen in an assay to detect autoantibodies specific to MUC1 thaneither MUC1 isolated from urine of normal individuals, synthetic MUC1 orMUC1 isolated from a range of different cultured cells. MUC1 isolatedfrom the serum of patients with advanced breast cancer is thereforepreferred for use as antigen in the panel assay method and the singlemarker assay methods described herein.

MUC1 has recently attracted interest as a target for immunotherapy ofadenocarcinomas and several Phase I clinical trials involving differentMUC1 vaccine substrates, adjuvants and carrier proteins have beencarried out (Goydos, J. S. et al. (1996) J Surgical Res. 63: 298-304;Xing, P. X. et al. (1995) Int. J. Oncol. 6: 1283-1289; Reddish, M. A. etal. (1996) Cancer Immunol. Immunother. 42: 303-309; Graham, R. A. et al.(1996) Cancer Immunol. Immunother. 42: 71-80). Methods for the detectionof anti-MUC1 autoantibodies using MUC1 isolated from the serum ofpatients with advanced breast cancer as antigen will be of particularuse in monitoring the success of MUC1 vaccine therapy. In this case theaim of the assay will be to detect anti-MUC1 antibodies produced inresponse to the vaccine rather than autoantibodies i.e. antibodiesproduced in response to an exogenous antigen introduced into the body byvaccination. Methods for the detection of autoantibodies directed toother tumour markers would also be of use in monitoring the success ofvaccine therapy using the relevant tumour marker. For example, followingvaccination with a p53 antigenic preparation, the presence of anti-p53antibodies could be monitored using the assay based on the use ofbiotinylated p53 antigen described in the examples given below.Moreover, the panel assay method could also be used in monitoring thesuccess of vaccine therapy, for example, in a situation where anindividual has been vaccinated with an antigenic preparation designed toelicit antibodies to two or more different tumour markers.

In a still further aspect the invention provides a method of detectingrecurrent disease in a patient previously diagnosed as carrying tumourcells, which patient has undergone treatment to reduce the number ofsaid tumour cells, which method comprises steps of contacting a sampleof bodily fluids from the patient with MUC1 protein or an antigenicfragment thereof, determining the presence or absence of complexes ofsaid MUC1 protein or antigenic fragment thereof bound to autoantibodiespresent in said sample of bodily fluids, said autoantibodies beingimmunologically specific to MUC1, whereby the presence of said complexesindicates the presence of recurrent disease in said patient.

The method described above may be repeated on a number of occasions toprovide continued monitoring for recurrence of disease. The method isparticularly preferred for the monitoring of patients previouslydiagnosed with primary breast cancer, colorectal cancer, prostate canceror bladder cancer, which patients have undergone treatment (e.g.surgery) to remove or reduce the size of their tumour. In this instance,the presence of anti-MUC1 autoantibodies in the patient's serum aftertreatment may be indicative of recurrence of disease.

Also provided by the invention are assay kits suitable for performingthe methods for the detection of autoantibodies described herein. Suchkits include, at least, samples of the tumour marker antigens to be usedas antigen in the assay and means for contacting the sample to be testedwith a sample of the antigen.

The contents of all documents, articles and references cited herein areincorporated herein by reference.

The present invention will be further understood with reference to thefollowing Examples and the accompanying Figures in which:

FIG. 1: shows the results of assays for autoantibodies to MUC1, p53 andc-erbB2 in samples of serum taken from 21 patients diagnosed withprimary breast cancer. Panel A: anti-p53 autoantibodies; Panel B:anti-c-erbB2 autoantibodies and Panel C: anti-MUC1 autoantibodies. Ineach case, the dotted line represents the cut-off value for normality.

FIG. 2: shows reactivity profiles of MUC1 protein isolated from normalhuman urine (panel A), ABC MUC1 isolated from the serum of patients withadvanced breast cancer (panel B) or MUC1 isolated from the human breastcancer cell line ZR75-1 (panel C) with various monoclonal anti-MUC1antibodies.

FIG. 3: shows continuous monitoring for recurrent disease in threepost-operative breast cancer patients. Quantitative assays foranti-MUC1, anti-c-erbB2 and anti-p53 autoantibodies and for the tumourmarker CA15-3 (TM) were performed on samples of serum taken at two orthree monthly intervals post-surgery.

FIG. 4: shows the range of autoantibody levels found in assays forautoantibodies to c-erbB2, c-myc, MUC1 and p53 in normal individuals andpatients with early primary breast cancer (PBC).

FIG. 5: summarises the detection rate for primary breast cancer in ananalysis of autoantibody levels in a series of healthy controls andpatients with primary breast cancer, PBC subdivided by Stage 1—i.e.lymph node negative and Stage 2—i.e. lymph node positive and patientswith metastatic cancer at 100% confidence.

FIG. 6: summarises the detection rate for primary breast cancer in ananalysis of autoantibody levels in a series of healthy controls andpatients with PBC subdivided by Stage 1—i.e. lymph node negative andStage 2—i.e. lymph node positive and patients with metastatic cancer at95% confidence.

FIG. 7: shows the sensitivity for primary breast cancer in an analysisof autoantibody levels in a series of healthy controls and patients withStage 1 or Stage 2 primary breast cancer at 95% confidence.

FIG. 8: shows the levels of autoantibodies to MUC1, p53 and c-erbB2 inthe serum of three patients previously diagnosed with breast cancermeasured sequentially during follow-up until the patient manifestedrecurrent disease.

FIG. 9: shows the autoantibody levels in further samples from the secondpatient in FIG. 10 (REC at 36 months) taken up to recurrence and duringtreatment for recurrence. Sequential measurements of established tumourmarkers reflecting tumour bulk (e.g. CA15-3 and CEA) were within thenormal range throughout this period (data not shown).

FIG. 10: shows follow-up autoantibody levels in post-operative serumsamples from two patients, one who did not develop recurrent disease (noREC) and the other who did (REC at 36 months).

FIG. 11: summarises the detection rates in an analysis of autoantibodylevels (p53, MUC1, c-erbB2 and c-myc) in samples of serum taken frompatients with urologically benign disorders and various stages ofbladder cancer.

* indicates patients which were benign with respect to urology (i.e. didnot have a urological malignancy), but six cases (all with positiveautoantibody status) had evidence of other malignancies.

** Other malignancies were:—lung cancer, skin cancer, adenocarcinoma ofunknown primary. Evidence of other neoplasia consisted of:—pleuraleffusion, ovarian cysts, colon polyps.

FIG. 12: summarises the detection rate for colorectal cancer in ananalysis of autoantibody levels in the serum of healthy controls,patients with colonic polyps and patients with colorectal cancer at 100%confidence compared to a pre-defined group of healthy controls.

FIG. 13: summarises the detection rate for colorectal cancer in ananalysis of autoantibody levels serum of healthy controls, patients withcolonic polyps and patients with colorectal cancer at at 95% confidencecompared to a pre-defined group of healthy controls.

FIG. 14: summarises the detection rate in an analysis of autoantibodylevels in the serum of healthy controls, patients with primary breastcancer and asymptomatic women known to be BRCA1 mutant carriers at 100%confidence compared to a pre-defined group of healthy controls.

FIG. 15: summarises the detection rate for prostate cancer in ananalysis of autoantibody levels in the serum of healthy controls andpatients with prostate cancer at 95% confidence compared to apre-defined group of healthy controls.

EXAMPLES Example 1 Isolation of ABC MUC1 from Advanced Breast CancerPatients

Method

ABC MUC1 was purified from pooled sera taken from 20 patients withadvanced breast cancer using immunoaffinity chromatography as follows:

The mouse monoclonal anti-MUC1 antibody B55 (also known as NCRC 11 anddescribed by Ellis et al. (1984) Histopathology. 8: 501-516 and inInternational patent application No. Wo 89/01153) was conjugated toCNBr-sepharose beads. Pooled sera from patients diagnosed with advancedbreast cancer was diluted 1/10 in phosphate buffered saline (PBS) andthen incubated with the antibody conjugated sepharose beads (25 mldiluted sera to 1 ml packed volume of beads) overnight at 4° C. withrolling. The beads were then packed by centrifugation and thesupernatant removed. In order to wash away unbound serum components thebeads were resuspended in PBS, rolled for 10 minutes, packed bycentrifugation and the supernatant removed. This washing sequence wasrepeated 5 times (or until A280 nm of the supernatant was ˜0). Thewashed beads were then resuspended in 0.25M glycine pH 2.5, rolled atroom temperature for 10 minutes, packed by centrifugation and thesupernatant removed. This supernatant was adjusted to pH 7 by theaddition of Tris and stored at 4° C. labelled ‘glycine fraction’. Thebeads were then resuspended in 1 ml 25 mM diethylamine (DEA) pH11,rolled at room temperature for 10 minutes, packed by centrifugation andthe supernatant removed. This supernatant was again adjusted to pH 7 bythe addition of Tris and stored at 4° C. labelled ‘25 DEA fraction’. Thebeads were finally resuspended in 1 ml 100 mM DEA pH11, rolled at roomtemperature for 10 minutes, packed by centrifugation and the supernatantremoved. The final supernatant was again adjusted to pH 7 by theaddition of Tris and stored at 4° C. labelled ‘100 DEA fraction’. TheMUC1 content of the three fractions (glycine fraction, 25 DEA fractionand 100 DEA fraction) was confirmed by ELISA using the mouse monoclonalanti-MUC1 antibody C595 (commercially available from Serotec).

Example 2 Immunological Characterisation of ABC MUC1 Isolated from theSerum of Patients with Advanced Breast Cancer

ABC MUC1 isolated from the serum at least 20 patients with advancedbreast cancer according to the procedure described in Example 1 can bedistinguished from MUC1 isolated from the urine of normal human subjects(normal human urinary MUC1) on the basis of altered affinity for thefollowing mouse monoclonal anti-MUC1 antibodies:

B55 (NCRC 11) C595 BC4W154 Obtainable from Hybritech, Inc DF3 Obtainablefrom Centocor B27.29 Obtainable from Biomira, Inc 115DB Obtainable fromCentocor 27.1 Obtainable from Austin Research Institute SM3 Obtainablefrom the Imperial Cancer Research Fund Ma552 Obtainable from CanAg HMPVObtainable from Austin Research Institute BC2 Obtainable from AustinResearch Institute

Normal urinary MUC1 is available from Dr M. R. Price, Cancer ResearchLaboratories, The University of Nottingham, University Park, Nottingham.NG7 2RD, United Kingdom.

The affinity of each of the above antibodies for ABC MUC1, normal humanurinary MUC1 and also MUC1 protein purified from the human breast cancercell line ZR75-1 (purified from a tissue culture supernatant by gelfiltration) was measured by performing colorimetric ELISA assays usingeach of the different antibodies and secondary anti-immunoglobulinantibodies conjugated to HRP. Following addition of the calorimetricsubstrate (TMB), measurements were taken of OD at 650 nm. The results ofthe ELISA assays are presented graphically in FIG. 2. Values of Kd forthe binding of several of these antibodies to ABC MUC1 and normal humanurinary MUC1 are summarised in Table 1:

TABLE 1 Kd values for binding of monoclonal antibodies to ABC MUC1 andnormal human urinary MUC1. Monoclonal Kd vs ABC MUC1 Kd vs urinary MUC1BC4W154 2.4 × 10⁻⁷  1.7 × 10⁻⁹ 115D8   1 × 10⁻⁸ 3.38 × 10⁻⁸ C595 2.4 ×10⁻⁸  2.5 × 10⁻⁸

Example 3 Cloning of Biotinylated p53

Method

Commercially available cDNA for p53 (E. coli clone pBH53, deposited inthe American Type Culture Collection under accession number 79110) wascloned into the PinPoint™ plasmid vector (Promega Corporation, MadisonWis., USA) using standard molecular biology techniques. The Pinpoint™vector is designed to facilitate the production of fusion proteinscomprising a biotinylation domain (consisting of a fragment of a biotincarboxylase carrier protein) fused N-terminally to the target protein ofinterest. Care was therefore taken during the cloning procedure toensure that the reading frame of p53 was maintained in the fusionprotein. Procedures for cloning in Pinpoint™ vectors are described indetail in the Promega Protocols and Applications Guide obtainable fromPromega Corporation, Madison Wis., USA.

Fusion proteins expressed from the PinPoint™ vector in E. coli arebiotinylated by an enzyme system of the E. coli host cells and maytherefore be purified or bound to an assay plate using conventionalavidin or streptavidin technology. For example, procedures forpurification of the fusion protein using avidin covalently attached to apolymethacrylate resin are described in the Promega Protocols andApplications Guide obtainable from Promega Corporation, Madison Wis.,USA.

Example 4 Cloning of c-erbB2

Method

Full-length cDNA encoding c-erbB2 was cloned from the human breastcancer cell line ZR75-1, which can be induced to up-regulate c-erbB2expression by treatment with the anti-cancer drug tamoxifen.

Two T25 flasks of sub-confluent ZR75-1 cells (available from theAmerican Type Culture Collection and from the European Collection ofCell Cultures, deposit number ATCC CRL1500) grown in RPMI plus 10%foetal calf serum were induced to express c-erbB2 by 4 day stimulationwith tamoxifen at 7.5 μM (see Warri et al. (1996) Eur. J. Cancer. 32A:134-140). The cells were then harvested using trypsin/EDTA and washedthree times with PBS.

mRNA was extracted from the cell pellet using a Dynabead mRNApurification kit according to the manufacturer's recommended protocol.The mRNA was then used as a template for first strand cDNA synthesisusing the Pharmacia Ready-to-go™ T primed first strand cDNA synthesiskit. cDNA/mRNA was then blunt end ligated into the EcoRV site of thePinPoint™ vector. The ligation products were then transformed into Top10 F E. coli cells (Invitrogen) following the manufacturer's suppliedprotocol and the transformed cells grown overnight on LB agar platescontaining ampicillin. Colonies of the transformed E. coli were copiedonto nitrocellulose filter and then grown for 2 hours on LB agarcontaining ampicillin and IPTG (1 mM). The colonies on thenitrocellulose filter were fixed and lysed (15 minutes in the presenceof chloroform vapour followed by 18 hours in 100 mM Tris/HCL pH 7.8; 150mM NaCl; 5 mM MgC12; 1.5% BSA; 1 μg/ml DNase 1; 40 μg/ml lysozyme).

Screening for colonies expressing anti-c-erbB2 reactive protein wascarried out as follows:

-   1. Wash nitrocellulose filter three times in TNT (10 mM Tris/HCl pH    8; 150 mM NaCl; 0.05% Tween 20) then block for 60 minutes in TNT+5%    dried milk protein.-   2. Incubate nitrocellulose filter for 2 hours at room temperature    with mouse anti-c-erbB2 antibody (Ab-3 from Oncogene Research    Products, Calbiochem)-   3. Wash the filter three times in TNT then incubate overnight at    4° C. with anti-mouse HRP conjugate.-   4. Wash filter three times in TNT, twice in TN (10 mM Tris/HCl pH 8;    150 mM NaCl) then visualise colonies expressing anti-c-erbB2    reactive protein using chloronaphthol (6 mg chloronaphthol in TN+6    μl 30% H₂O₂)-   5. After development (approximately 20 minutes treatment with    chloronaphthol as described in step 4) wash filter with water and    allow to air dry.

Colonies identified as positive for c-erbB2 expression were picked andgrown up overnight in liquid culture of LB+ampicillin and small amountsof plasmid DNA and protein were prepared from the culture for analysis.Plasmids containing a c-erbB2 cDNA insert were identified usingrestriction enzyme digestion and PCR using a primer pair specific to thepublished c-erbB2 cDNA sequence, described by Yazici, H. et al. (1996)Cancer Lett. 107: 235-239. DNA sequence analysis could then be used toconfirm 1) the presence of a c-erbB2 insert and 2) that the readingframe of c-erbB2 is maintained in the resultant biotinylated fusionprotein. Protein samples prepared from E. coli cultures carrying aplasmid with a c-erbB2 insert were analysed by SDS-PAGE and westernblotting to ensure that the correct protein was being expressed.

Example 5 Detection of the Immune Response of Patients with PrimaryBreast Cancer Using a Panel Assay

Methods:

(A) Preparation of Biotinylated Antigen

E. coli transformed with the appropriate PinPoint™ plasmid expressingbiotinylated antigen were grown in a 5 ml overnight culture(LB+amp+biotin) and the overnight culture used to inoculate a 150 mlculture. The 150 ml culture was grown to OD 0.4-0.6 then expression ofthe fusion protein was induced by the addition of IPTG to a finalconcentration of 1 mM and the induced culture incubated at 25° C. Thebacterial cells were harvested by centrifugation and then lysed bygentle sonication in a Tris/EDTA buffer containing the proteaseinhibitor PMSF. Cellular debris was removed by centrifugation at ˜50,000g and the resultant particle-free supernatant assayed by avidin ELISA toconfirm the presence of biotinylated protein.

(B) c-erbB2/p53 Autoantibody Assay Method

-   1. Standard 96 well microtitre assay plates were coated with avidin,    using 50 μl of a 1 μg/ml solution per well, and allowed to air dry    overnight. The plates were then washed once with PBS/Tween to remove    residual salt crystals, blocked for 60 minutes with a solution of 2%    (w/v) PVP (polyvinylpyrolidone 360) in PBS and washed three times    using PBS/Tween.-   2. Particle free supernatant containing the appropriate biotinylated    antigen (prepared as described in section (1) above) was plated out    at 50 μl per avidin-coated well and then incubated for 60 minutes at    room temperature with shaking to allow the biotin/avidin binding    reaction to take place. The plates were then washed four times with    PBS/Tween.-   3. Serum samples to be tested for the presence of autoantibodies    (diluted 1/50 and 1/100 in PBS) were plated out in triplicate (50 μl    per well) and then incubated for 60 minutes with shaking to allow    formation of any autoantibody/antigen complexes. Plates were then    washed four times with PBS/Tween to remove unbound serum components.-   4. 50 μl of HRP conjugated anti-human IgG/IgM antibody (obtained    from Dako and used at a dilution recommended by the manufacturer)    was added to each well and incubated for 60 minutes at room    temperature with shaking. The plates were then washed again four    times with PBS/Tween.-   5. 50 μl of TMB was added to each well and measurements of OD at 650    nm for each well of the assay plate were taken kinetically over a    period of 10 minutes.

For each antigen, control assays were performed following the proceduredescribed above but using a sample of protein induced from E. colitransformed with a control PinPoint™ vector containing an out-of-framecDNA instead of the particle free supernatant containing biotinylatedantigen. As it will be apparent to persons skilled in the art, the abovemethodology can be adapted for use in the detection of autoantibodies ofany specificity with use of an appropriate biotinylated antigen.

(C) MUC1 Autoantibody Assay

-   1. ABC MUC1 isolated from the serum of patients with advanced breast    cancer according to the method of Example 1 (all three fractions    pooled) was diluted appropriately in PBS, plated out on a 96 well    microtitre assay plate at 50 μl per well and left to dry overnight.    The plate was then washed once with PBS/Tween to remove residual    salt crystals, blocked for 60 minutes using a solution of 2% (w/v)    PVP in PBS and washed three times with PBS/Tween.-   2. Serum samples to be tested for the presence of autoantibodies    (diluted 1/50 and 1/100 in PBS) were plated out in triplicate,    adding 50 μl per well, and incubated for 60 minutes at room    temperature with shaking. The plate was then washed four times with    PBS/Tween.-   3. 50 μl of HRP conjugated anti-human IgG/IgM antibody (obtained    from Dako and used at a dilution recommended by the manufacturer)    was added to each well and incubated for 60 minutes at room    temperature with shaking. The plates were then washed again four    times with PBS/Tween.-   4. 50 μl of TMB was added to each well and measurements of OD at 650    nm for each well of the assay plate were taken kinetically over a    period of 10 minutes.    Results

Pre-operative blood samples taken from 21 patients diagnosed withprimary breast cancer were assayed for the presence of autoantibodiesagainst MUC1, p53 and c-erbB2. The results of these assays are shown inFIG. 1 and summarised in Table 2, on page 30.

TABLE 2 Sample anti-p53 Prediction anti-c-erbB2 Prediction anti-MUC1Prediction Combined 1 + cancer − normal + cancer CANCER 2 +/− ? +/− ?+/− ? cancer 3 + cancer +/− ? + cancer CANCER 4 + cancer + cancer +cancer CANCER 5 + cancer + cancer +/− ? CANCER 6 − normal + cancer +/− ?cancer 7 + cancer + cancer + cancer CANCER 8 +/− ? + cancer +/− ? CANCER9 + cancer + cancer + cancer CANCER 10 + cancer + cancer − normal CANCER11 +/− ? + cancer + cancer CANCER 12 − normal + cancer − normal cancer13 + cancer − normal + cancer CANCER 14 +/− ? + cancer + cancer CANCER15 + cancer − normal + cancer CANCER 16 − normal − normal +/− ? ? 17 +/−? − normal + cancer cancer 18 + cancer + cancer + cancer CANCER 19 +cancer + cancer + cancer CANCER 20 + cancer − normal + cancer CANCER21 + cancer +/− ? − normal cancer

FIG. 1 shows the results of the assays for autoantibodies specific toMUC1, c-erbB2 and p53. For each set of data the dotted line representsthe cut-off value for normality. For the purposes of this study thenormal control patients were women who clinically and/ormammographically had no evidence of breast cancer at the time of takingthe serum sample. In order to establish the cut-off value for normality,control assays were performed on a total of 30 normal patients. Valuesbelow the dotted line fall within the normal control range and werescored as negative (−) in Table 2 whereas values above the dotted linewere scored as positive (+). Values which were difficult to score asnegative or positive with a reasonable degree of certainty were scored+/−. Patients scoring positive in at least two of the assays wereidentified as strongly positive for breast cancer (indicated “CANCER” inTable 2); patients scoring positive in at least one of the assays wereidentified as probable for breast cancer (indicated “cancer” in Table2).

The results presented illustrate the predictive value of the threeautoantibody assays both when used individually and when used as apanel. The use of a single assay to predict breast cancer gaveapproximately 40% of the results as a false negatives. However, bycombining the results from all three assays only one patient appeared asa false negative (<5%), 71% of patients were scored as strongly positivefor breast cancer (i.e. positive in at least two assays) and 23% ofpatients were scored as probable for breast cancer (i.e. positive in atleast one assay). The results also show that a group of patients whichhave all been diagnosed with primary breast cancer have differentserological profiles in terms of the immune response to their cancer.Thus, no single one of the three autoantibody assays would be useful inall primary breast cancer patients.

Example 6 Cloning of a Ras Antigen

Method

cDNA encoding a mutant oncogenic form of ras (designated K-ras) wascloned from the cell line KNRK (Rat kidney, Kirsten MSV transformed, seeAaronson, S. A. and Weaver, C. A. (1971) J. Gen. Virol. 13: 245-252;ATCC accession number CRL 1569). mRNA was extracted from the cell pelletusing a Dynabead mRNA purification kit according to the manufacturer'srecommended protocol. cDNA synthesis, cloning into the EcoRV site of thePinPoint™ vector and transformation of E. coli was carried out asdescribed in Example 4. Clones expressing ras were then identified byexpression screening using the anti-ras antibody F234-4.2 fromCalbiochem.

Example 7 Cloning of c-myc Method

cDNA encoding human c-myc was cloned from the breast cancer cell lineT47-D (European Collection of Animal Cell Cultures accession number85102201). mRNA was extracted from the cell pellet using a Dynabead mRNApurification kit according to the manufacturer's recommended protocol.cDNA synthesis, cloning into the EcoRV site of the PinPoint™ vector andtransformation of E. coli was carried out as described in Example 4.Clones expressing c-myc were then identified by expression screeningusing the anti-c-myc antibody 4111.1 from Unilever.

Example 8 Assay for Ras and c-myc Autoantibodies

Biotinylated c-myc and ras antigens were prepared from E. colitransformed with the appropriate PinPoint™ plasmid vector expressingbiotinylated c-myc or biotinylated ras, as described in Example (5),part (A). The assays for c-myc and ras autoantibodies were thenperformed according to the protocol described in Example (5), part (B).

Example 9 Method of Detecting Recurrent Disease in a Patient PreviouslyDiagnosed as Carrying Tumour Cells

A group of nine patients previously diagnosed with primary breast cancerwere selected. Pre-operative serum samples were taken from each of thesepatients prior to surgery for the removal of the primary breast cancer.Follow-up serum samples were then taken postoperatively at 2 or 3monthly intervals and during the same period of time the patients wereassessed clinically for signs of recurrent disease. None of the patientsreceived any post-operative therapy until recurrence was diagnosedclinically. The preoperative and post-operative serum samples from eachof the patients were assayed for the presence of autoantibodies to MUC1,c-erbB2 and p53, using the assay methods described above under Example5, and also for the presence of the commonly used serum tumour markerprotein CA15-3. The results of these assays are summarised in Table 3,on pages 35 and 36 and results for three of the nine patients arepresented graphically in FIG. 3.

Clinical signs of recurrent disease were scored as follows:

LN recurrent disease in the lymph nodes

LR local recurrence

METS distant metastases present

Results

In each of the patients at least one class of autoantibody was observedto remain above normal level. This suggests continued presence of thetumour marker (immunogen) and hence continued presence of tumour. Serumlevels of the tumour marker protein CA15-3 were not found to bepredictive of recurrent disease.

TABLE 3 Anti- Pre- Anti Pre- Anti Pre- Recur- Date of first DFI PatientSample date CA 15-3 p53 diction c-erbB2 diction MUC1 diction Predictedrence recurrence (months) 0001 December 1988 11 − + Cancer + CancerCANCER − March 1987 12 − + Cancer + Cancer CANCER − May 1987 13 − +Cancer + Cancer CANCER − August 1987 22 +/− ? + Cancer + Cancer CANCER −November 1987 56 +/− ? + Cancer + Cancer CANCER METS December 1987 79+/− ? + Cancer + Cancer CANCER METS 11 0002 January 1987 16 − + Cancer+/− ? Cancer − May 1987 8 − + Cancer +/− ? Cancer − August 1987 10 − +Cancer + Cancer CANCER − November 1987 12 +/− ? + Cancer + Cancer CANCER− February 1988 16 − + Cancer + Cancer CANCER − February 1989 23 0003February 1987 10 − + Cancer − Cancer − May 1987 7 + Cancer + Cancer −CANCER − August 1987 8 + Cancer + Cancer − CANCER − November 1987 12 +Cancer + Cancer − CANCER − February 1988 12 + Cancer + Cancer − CANCER −May 1988 11 − + Cancer − Cancer − December 1989 34 0004 February 19878 + Cancer ++ Cancer − CANCER − April 1987 + Cancer + Cancer − CANCER −June 1987 4 + Cancer + Cancer − CANCER − December 1987 0.4 + Cancer ++Cancer − CANCER − March 1988 7 ++ Cancer ++ Cancer − CANCER − February1993 71 0005 March 1987 16 +/− ? + Cancer − Cancer − June 1987 13 +/−? + Cancer − Cancer − September 1987 14 + Cancer + Cancer +/− ? CANCER −December 1987 17 +/− ? + Cancer +/− ? CANCER − March 1988 16 − May 1988LN 15 0006 May 1987 12 − + Cancer + Cancer CANCER − July 1987 15 − +Cancer + Cancer CANCER − September 1987 9 +/− ? + Cancer +/− ? Cancer LR4 November 1987 12 − + Cancer +/− ? Cancer − March 1988 15 − +/− ? − −May 1988 13 − +/− ? − − November 1988 000& June 1987 26 + Cancer ++Cancer − CANCER − August 1987 28 + Cancer + Cancer − CANCER − October1987 42 + Cancer + Cancer − CANCER − December 1987 105 + Cancer ++Cancer + Cancer CANCER METS December 1987 6 0008 June 1987 48 + Cancer +Cancer + Cancer CANCER − August 1987 30 + Cancer + Cancer + CancerCANCER − October 1987 17 + Cancer + Cancer + Cancer CANCER − January1988 14 + Cancer + Cancer + Cancer CANCER − May 1988 22 + Cancer +Cancer +/− ? CANCER LR May 1988 11 0009 May 1987 17 − +/− ? − − August1987 17 − + Cancer − Cancer − November 1987 18 − + Cancer − Cancer LR 6January 1988 31 − + Cancer +/− ? Cancer METS 8

Example 10 Retrospective Analysis of a Well Characterised Series ofHealthy Controls and Patients with Early Breast Cancer

The above-described methods for detecting autoantibodies to MUC1, p53,c-erbB2 and c-myc were used to carry out a retrospective study on alarge number of early (stage 1 and 2) breast cancer sera as well as alarge number of control serum samples from individuals with no evidenceof malignancy (control group). The serum samples from patients were alltaken within a 4 week pre-operative period. At the same time, the serumsamples were assayed for the presence of circulating antigen (MUC1 andc-erbB2) using conventional tumour marker kits (used normally inadvanced disease only). This would allow an assessment of whether theautoantibody assays are more sensitive than the conventional antigenassays. As used herein, the terms early or primary breast cancer meansthat the primary tumour has a diameter of less than 5 cm. Stage 1 earlybreast cancer is defined as lymph node negative; Stage 2 early breastcancer is defined as lymph node positive.

In total, pre-operative serum samples from 200 patients diagnosed withprimary breast cancer and 100 normal control samples were assayed forautoantibodies against MUC1, p53, c-erbB2 and c-myc. The results aresummarised in Tables 4-7 and FIGS. 4-7.

FIG. 4 depicts the range of autoantibody levels found for each assay innormal individuals and patients with early breast cancer. It is apparentthat cancer patients have a considerably higher level of circulatingautoantibodies to these markers than do normal individuals. Using therange for the normal individuals it is possible to set a ‘cut-off’ abovewhich no normal values should lie. Therefore, samples with autoantibodylevels above this cut-off can be deemed to be positive for cancer.Cut-off points determined in this manner were used to score the resultsof the retrospective study in early breast cancer patients.

The results presented in Tables 4-7 and FIGS. 5-7 demonstrate thepredictive value of the four autoantibody assays both individually andwhen used in combination as a panel of assays. Table 4 indicates theincreased sensitivity of combining the results of a number of assays. Byusing one assay on its own, less than 50% of cancers are detected,however the power of detection increases as more assays are added to thepanel until the combination of all four assays allows 82% of primarycancers to be detected. FIG. 7 shows the percentage of samples which arepositive in 0 out of 4 assays up to 4 out of 4 assays. This providesgood evidence that the panel assay is more powerful in the detection ofcancer than any one single marker assay since not all patients withcancer have raised autoantibodies to all markers.

Tables 5-7 summarise the detection rates in stage 1, stage 2 and inearly breast cancer (i.e. stage 1 and 2) for various combinations ofautoantibody assays. The use of a single autoantibody assay to predictbreast cancer gives approximately 60-70% of the results as falsenegatives in the stage 1 group; and 50-60% in stage 2. However, bycombining the results from all four assays, 76% of stage 1 and 89% ofstage 2 cancers were positive in one or more assay. The overalldetection rate for early breast cancer (i.e. both stage 1 and stage 2cancers) using this system was 82%. In both stage 1 and stage 2 cancer,assaying for autoantibodies to MUC1 appeared to add predictive power toany combination of assays.

The results for this study were obtained using a 100% confidence limit,in other words for a result to be deemed positive it had to fall abovethe cut-off for readings in the normal range. This normal range waspreviously evaluated from a large number of normal individuals and thenconfirmed using the control group of 100 normal individuals mentionedabove. Therefore, within the normal control group, none of the sampleswere found to be positive, meaning that the sensitivity of the panel ofautoantibody assays was 100% for the detection of early breast cancer(FIG. 5).

FIGS. 6 and 7 demonstrate the detection rates which are achievable ifspecificity is reduced from a 100% confidence level (no false positives)to a 95% confidence level, where some degree of false positive detectionis expected. In this case, the cut-off point is defined as the meanvalue plus twice the standard deviation of the normal sample range.Using this cut-off point, approximately 5% of the normal samples weredetermined to be positive for cancer (i.e. false positives); whilstdetection of primary cancer increased to approximately 94% (i.e. 6%false negatives). Again, the greatest percentage of the sample groupwere positive in only 1 out of the 4 assays, however, the percentage ofsamples that were positive in all 4 assays increased considerably.

Since the above study was carried out retrospectively, clinical data wasavailable regarding the initial diagnosis as well as clinical dataregarding the post-operative outcome (i.e. follow-up data). This allowedanalysis of the prognostic value of the data obtained from theautoantibody assays. Table 8 shows the correlations between serum levelsof autoantibodies to MUC1, p53, c-erbB2 and c-myc and a number ofclinical factors. For instance, the presence of autoantibodies to any ofthe 4 tumour associated proteins (MUC1, p53, c-erbB2 or c-myc) appearsto correlate with the development of a recurrence. In other words, thosepatients who had autoantibodies were more likely to go on to develop arecurrence of their disease. In the case of autoantibodies to MUC1,c-myc and c-erbB2, this was most likely to be distant metastases, onlyautoantibodies to p53 were not associated with the later development ofdistant metastases with any statistical significance. In fact, thepresence of autoantibodies to p53 was the weakest indicator of a laterrecurrence of disease; furthermore, p53 autoantibodies correlated withdisease free interval.

Table 9 presents an analysis of whether the degree of autoantibodypositivity may be of value in the prediction of which stage 1 tumourwill go on to develop a recurrence. At the present time, there is littleto indicate at the time of diagnosis whether a patient with a stage 1tumour (i.e. no evidence of spread of tumour to the lymphatic system)will go on to develop recurrent disease. As can be seen in Table 9, ofthose patients with stage 1 tumours from the sample group that went onto develop recurrent disease, 71% were positive in two or moreautoantibody assays. Of the patients with stage 1 tumours that have notyet recurred, only 30% were positive in two or more autoantibody assays.

TABLE 4 Sensitivity of autoantibody assays in the detection of earlybreast cancer. % PBC positive Single marker assay 35-47 Two marker assay51-60 Three marker assay 63-76 Four marker assay 82

TABLE 5 Sensitivity of autoantibody panel assays in the detection ofstage 1 breast cancer. p53 c-erbB2 c-myc MUC1 p53 38 48 58 59 c-erbB2 3150 51 c-myc 41 55 MUC1 38 p53/c-erbB2 61 66 p53/c-myc 73 c-erbB2/c-myc65 p53/c-erbB2 /c-myc 76

TABLE 6 Sensitivity of autoantibody panel assays in the detection ofstage 2 breast cancer. p53 c-erbB2 c-myc MUC1 p53 40 56 55 73 c-erbB2 4256 73 c-myc 33 69 MUC1 56 p53/c-erbB2 65 84 p53/c-myc 80 c-erbB2/c-myc84 p53/c-erbB2/c-myc 89

TABLE 7 Sensitivity of autoantibody panel assays in the detection ofprimary breast cancer. p53 c-erbB2 c-myc MUC1 p53 38 51 57 64 c-erbB2 3553 59 c-myc 37 60 MUC1 47 p53/c-erbB2 63 73 p53/c-myc 76 c-erbB2/c-myc72 p53/c-erbB2/c-myc 82

TABLE 8 Correlations between serum autoantibody level and variousclinical factors. FACTOR MUC1 p53 c-erbB2 c-myc recurrence ✓ 1✓4 ✓ ✓local recurrence 1✓2 1✓2 1✓2 1✓4 distant metastases ✓ X ✓ ✓ stage X X XX grade X X X X family history X X X X disease free interval X ✓ X X ageX X X X menopausal status X X X X Key: ✓ Good correlation 1✓2 Moderatecorrelation 1✓4 Weak correlation X No correlation

TABLE 9 Analysis of the degree of positivity in autoantibody assays forrecurrent and nonrecurrent stage 1 breast cancer tumours. Negative-no+ve auto- +ve auto autoantibodies antibodies to antibodies to detectedone marker 2-4 markers Recurrent 12% 17% 71% Non-recurrent 22% 48% 30%

Example 11 Detection of Autoantibodies in Sequential SerumSamples-Application to the Monitoring of Disease Progression

This study was carried out in order to assess whether autoantibodyassays could be useful in the earlier detection of recurrent disease.

Levels of autoantibodies to MUC, p53 and c-erbB2 in the serum ofpatients previously diagnosed with breast cancer were measuredsequentially during follow-up until the patient manifested recurrentdisease. The results are summarised in FIGS. 8-10. All three patientswent on to develop recurrent disease. In all three patients,autoantibody levels were indicative of the presence of cancer. However,there is no evidence from this group that autoantibody levels decreaseafter removal of the primary tumour. FIG. 10 shows the levels ofautoantibodies post-operatively of a patient with non-recurrent diseaseand a patient with recurrent disease. Autoantibody levels in the patientwith non-recurrent disease remained below the cut-off point during theperiod of sample collection (48 months). In the second patient, whosedisease recurred at 36 months, autoantibody levels are seen to besteadily rising towards the cut-off point, with c-erbB2 autoantibodiesrising above cut-off. Furthermore, as can be seen in FIG. 9, whenfurther sequential samples are added to the analysis, 3 out of the 4assays become positive for cancer and these levels then decrease againonce treatment of the recurrence is underway. This data supports theutility of autoantibody assays in the earlier detection of recurrentdisease.

Example 12 Analysis of a Series of Patients with Bladder Cancer andBenign Urological Disorders

Serum samples were collected from a group of 80 patients with bladdercancer/benign urological disorders and analysed for the presence ofautoantibodies to MUC1, p53, c-erbB2 and c-myc using the assay methodsdescribed above.

The data summarised in Table 10 shows that single assay sensitivitiesfor bladder cancer detection range from 15-50% (as opposed to 35-47% forbreast cancer). The detection sensitivity using all 4 assays was 80%,similar to that found for early breast cancer.

FIG. 11 shows the break down of detection rates between urologicallybenign disorders (‘benign’) and the three stages of bladder cancer. Uponfurther investigation of the relevant clinical data it became apparentthat 6 of the patients in the ‘benign’ group had evidence of othermalignancies. These other malignancies were lung cancer, skin cancer andadenocarcinoma. Evidence of other malignancies were: pleural effusion,ovarian cysts and colon polyps. Serum samples from all 6 of thesepatients had been scored as positive for cancer using the panel ofautoantibody assays, illustrating the general application of the panelassay to the detection of cancers. Furthermore, it is known that somepatients with stage PT1/2 and PT3/4 disease had previously receivedsystemic therapy.

TABLE 10 Sensitivity of autoantibody assays in the detection of bladdercancer. % positive Single marker assay 15-50 Two marker assay 28-73Three marker assay 46-76 Four marker assay 80

TABLE 11 Sensitivity of autoantibody panel assays in the detection ofbladder cancer. p53 c-erbB2 c-myc MUC1 p53 50 73 73 73 c-erbB2 17 28 36c-myc 15 35 MUC1 24 p53/c-erbB2 76 76 p53/c-myc 75 c-erbB2/c-myc 46p53/c-erbB2/c-myc 80

Example 13 Sensitivity of Autoantibody Assay in Diagnosis of ColorectalCancer

An autoantibody assay as previously described was carried out on serumsamples from patients with colorectal cancer using the tumour antigensc-myc, p53, c-erbB2 and K-ras individually and as a panel. The resultsare shown in FIGS. 12 and 13. As has been demonstrated previouslyincreased sensitivity is shown when a panel of antigens is used.

Example 14 Use of BRCA1 in Panel Assay for Detection of Breast Cancer

A BRCA1 antigen suitable for use in the detection of anti-BRCA1autoantibodies was cloned from the breast cancer cell line MCF7 using anRT-PCR strategy. Briefly, mRNA isolated from MCF7 cells was reversetranscribed to give first-strand cDNA. These cDNA was used as a templatefor PCR using a primer pair designed to amplify a product covering thefirst 1500 base pairs of the BRCA1 cDNA but including a known mis-matchmutation that leads to an early stop codon and therefore the productionof truncated protein. Different sites for restriction enzyme digestionwere also incorporated into the forward and reverse PCR primers tofacilitate the cloning of the PCR product. The PCR primers were asfollows:

5′-GAC AGG ATC CGG ATG GAT TTA TCT GCT CTT CGC GTT G (SEQ. ID. NO. 1)

5′-GCG GCC GCC CTC ATG TAG GTC TCC TTT TAC GC (SEQ. ID. NO. 2)

The PCR product obtained using these primers was then cloned into thePinPoint™ vector and used to transform E. coli Top 10 F cells, asdescribed hereinbefore. Clones expressing the fusion protein oftruncated BRCA1 antigen fused in-frame to the N-terminal biotinylationdomain were then identified by expression screening, according to theprocedure described in Example 4, using the antibody MAB4132 fromChemicon.

Biotinylated truncated BRCA1 antigen is then prepared from E. colitransformed with the appropriate PinPoint™ plasmid vector expressing thefusion protein, as described in Example (5), part (A). The assay forBRCA1 autoantibodies is then performed according to the protocoldescribed in Example (5), part (B).

FIG. 14 shows the results of a study in which the above-described assaysfor autoantibodies to c-myc, p53, c-erbB2, MUC1 and BRCA1 were performedindividually, as a panel and as a panel without BRCA1 to detectautoantibodies in samples of serum taken from normal individuals,patients diagnosed with primary breast cancer and BRCA1 mutationcarriers. As demonstrated previously, increased sensitivity is shownwhen a panel of markers is used.

Example 15 Use of Autoantibody Panel Assay for Detecting ProstateCancer, Incorporating PSA

cDNA encoding human PSA was cloned from the cell line T47-D using aprotocol similar to that described above for the cloning of c-erbB2.Briefly, the T47-D cells were first stimulated with Apigenin at 10-5M asdescribed by Rosenberg et al. (1998) Biochem Biophys Res Commun. 248:935-939. mRNA was then extracted and cDNA synthesis, ligation intoPinpoint™ and transformation of E. coli. performed as described inExample 4. Clones expressing PSA were identified using an anti-PSAantibody. Biotinylated PSA antigen was prepared from E. coli transformedwith the PinPoint™ vector expressing biotinylated PSA according to theprotocol described in Example (5), part (A). The assay for PSAautoantibodies was then performed according to the protocol described inExample (5), part (B).

An autoantibody assay using the methods described above was carried outon patients with prostate cancer using c-myc, p53, c-erbB2, PSA and MUC1 individually and as a panel. The results are shown in FIG. 15 andconfirm the increased sensitivity of such a panel for detection ofprostate cancer.

Example 15 Other Tumour Marker Antigens

CA125 can be affinity purified from the ovarian cancer cell lineOVRCAR-3 (available from the ATCC) using Mab VK-8, as described byLloyd, K. O. et al. (1997) Int. J. Cancer. 71: 842-850.

APC protein is expressed by the colorectal cancer cell line SW480(available from the ATCC) as described by Munemitsu, S. et al. (1995)PNAS 92: 3046-3050.

1. A method for the detection of cancer or early neoplastic change,comprising (a) contacting a sample of bodily fluids from a mammal with apanel of two or more distinct tumor marker antigens; and (b) determiningthe presence or absence of complexes of the tumor marker antigens boundto autoantibodies present in the sample of bodily fluids, theautoantibodies being immunologically specific to tumor marker proteins;wherein the presence of the complexes is indicative of cancer or earlyneoplastic change, wherein the panel provides higher sensitivity and/orspecificity than a single tumor marker antigen, and wherein at least oneof the two or more tumor marker antigens is selected from the groupconsisting of MUC1, p53, c-erbB2, Ras, and c-myc.
 2. A method for thedetection of cancer or early neoplastic change, comprising: (a)contacting a sample of bodily fluids from a mammal with a panel of twoor more distinct tumor marker antigens; and (b) determining the presenceor absence of complexes of the tumor marker antigens bound toautoantibodies present in the sample of bodily fluids, theautoantibodies being immunologically specific to tumor marker proteins;wherein the presence of the complexes is indicative of cancer or earlyneoplastic change, wherein the complexes detected are indicative ofcancer, wherein the panel provides higher sensitivity and/or specificitythan a single tumor marker antigen, and wherein the panel comprises atleast p53 and c-erbB2.
 3. The method of claim 2 wherein the cancer isbladder cancer and the panel also includes at least one tumor markerantigen selected from the group consisting of MUC1 and c-myc.
 4. Themethod of claim 2 wherein the cancer is breast cancer and the panel alsoincludes MUC1.
 5. A method for the detection of cancer in asymptomaticpatients or early neoplastic change, comprising (a) contacting a sampleof bodily fluids from a mammal with a panel of two or more distincttumor marker antigens; and (b) determining the presence or absence ofcomplexes of the tumor marker antigens bound to autoantibodies presentin the sample of bodily fluids, the autoantibodies being immunologicallyspecific to tumor marker proteins; wherein the presence of the complexesis indicative of cancer or early neoplastic change, wherein the panelprovides higher sensitivity and/or specificity than a single tumormarker antigen, and wherein the cancer is an early carcinogenic changein asymptomatic patients.
 6. A method for the detection of cancer orearly neoplastic chance, comprising (a) contacting a sample of bodilyfluids from a mammal with a panel of two or more distinct tumor markerantigens; and (b) determining the presence or absence of complexes ofthe tumor marker antigens bound to autoantibodies present in the sampleof bodily fluids, the autoantibodies being immunologically specific totumor marker proteins; wherein the presence of the complexes isindicative of cancer or early neoplastic change, wherein the panelprovides higher sensitivity and/or specificity than a single tumormarker antigen, and wherein the tumor marker proteins have alterationsthat are absent in corresponding normal tissue proteins.
 7. A method forthe detection of cancer or early neoplastic change, comprising (a)contacting a sample of bodily fluids from a mammal with a panel of twoor more distinct tumor marker antigens; and (b) determining the presenceor absence of complexes of the tumor marker antigens bound toautoantibodies present in the sample of bodily fluids, theautoantibodies being immunologically specific to tumor marker proteins;wherein at least one of the two or more tumor marker antigens isselected from the group consisting of MUC1, p53, c-erbB2, Ras, c-myc,BRCA1, BRCA2, PSA, APC and CA125 and wherein the presence of thecomplexes is indicative of cancer or early neoplastic change.
 8. Themethod of claim 7 wherein at least one of the two or more tumor markerantigens is labelled with a protein or peptide tag.
 9. The method ofclaim 7 wherein at least one of the two or more tumor marker antigens islabelled with biotin.
 10. The method of claim 7 wherein the complexesdetected are indicative of cancer.
 11. The method of claim 10 whereinthe panel comprises at least p53 and c-erbB2.
 12. The method of claim 11wherein the cancer is bladder cancer and the panel also includes atleast one tumor marker antigen selected from the group consisting ofMUC1 and c-myc.
 13. The method of claim 11 wherein the cancer iscolorectal cancer and the panel also includes at least one tumor markerantigen selected from the group consisting of Ras and APC.
 14. Themethod of claim 11 wherein the cancer is prostate cancer and the panelalso includes at least one tumor marker antigen selected from the groupconsisting of PSA and BRCA1.
 15. The method of claim 11 wherein thecancer is breast cancer and the panel also includes MUC1.
 16. The methodof claim 11 wherein the cancer is ovarian cancer and the panel alsoincludes at least one tumour marker antigen selected from the groupconsisting of BRCA1 and CA125.
 17. The method of claim 10 wherein thecancer is colorectal cancer and the panel is selected from at least twoof p53, Ras, c-erbB2 and APC.
 18. The method of claim 10 wherein thecancer is prostate cancer and the panel is selected from at least two ofp53, PSA, BRCA1 and c-erbB2.
 19. The method of claim 10 wherein thecancer is ovarian cancer and the panel is selected from at least two ofp53, CA125, c-erbB2 and BRCA1.
 20. The method of claim 10 wherein thecancer is breast cancer and the panel is selected from at least two ofp53, MUC1, c-erbB2, c-myc, BRCA1, BRCA2 and PSA.
 21. The method of claim7 wherein the cancer is an early carcinogenic change in asymptomaticpatients.
 22. The method of claim 7 wherein the cancer is recurrentdisease in a patient previously diagnosed as carrying tumor cells, whichpatient has undergone treatment to reduce the number of the tumor cells.